Molecular mechanism of hematopoietic regulation by leukemia-related transcription factor AML1(PEBP2αB)

本研究利用本课题新建立的体外实验系统，分析了白血病相关转录因子AML 1（PEBP 2 αB）在造血调控中的分子作用机制。主要研究结果如下：1.我们建立了一个利用小鼠胚胎干细胞（ES细胞）分化的体外实验系统，通过该系统可以在体外复制AML 1缺陷动物体内观察到的血液学表型.由AML 1纯合无效等位基因引起的造血缺陷可以通过重新表达野生型AML 1 cDNA来挽救，这表明令人信服的证据表明AML 1敲除表型仅仅是由于该基因的缺失。在体内观察到这种拯救，因为拯救克隆有助于嵌合体小鼠中的淋巴造血。该拯救需要AML1分子的反式激活结构域.相同的AML1 cDNA的强制表达不能挽救造血缺陷，这表明AML1的转录控制对于PEBP 2转录复合物的生物活性是重要的。与这一观察结果相一致，AML 1的表达水平随着ES细胞的体外分化而波动。我们分析了已知的AML1靶基因的表达，发现即使在没有活性AML1分子的情况下，它们中的大多数也被保留下来。因此，这表明存在尚未鉴定的转录靶点，其介导AML 1的生物活性。

We analyzed the molecular mechanism of actions played by leukemia-related transcription factor, AML1(PEBP2αB), on hematopoietic regulation by using an in vitro experimental system, which was newly established through this research project. Our results are summarized as follows :1. We established an in vitro experimental system by using murine embryonic stem(ES) cell differentiation, through which the in vivo hematological phenotype observed in the AML1-deficient animals could be replicated in vitro.2. The hematopoietic defect resulting from homozygous null allele for AML1 could be rescued by re-expressing wild-type AML1 cDNA, indicating compelling evidence that the AML1-knockout phenotype is due solely to the lack of this gene.3. This rescue was observed in vivo in that the rescue clones contribute to lympho-hematopoiesis in chimera mice.4. The rescue required the transactivation domain of AML1 molecule.5. Forced expression of the same AML1 cDNA did not rescue the hematopoietic defect, suggesting that transcriptional control of AML1 was important for the biologic activity of the PEBP2 transcription complex. Consistent with this observation, the expression level of AML1 fluctuated as ES cells differentiated in vitro.6. We analyzed the expression of the known AML1-target genes and found that most of them were retained even in the absence of the active AML1 molecule. Therefore, it is suggested that as yet un-identified transcriptional target(s) exists which mediates AML1's biologic activities.